BHLH112 Antibody

What is bHLH112 and why is it important in plant research?

bHLH112 belongs to the basic helix-loop-helix transcription factor family, one of the largest transcription factor families in plants. These proteins contain a highly conserved bHLH domain consisting of a DNA-binding basic region and a protein-interaction helix-loop-helix region. bHLH112 plays critical roles in plant responses to abiotic stresses, particularly drought, cold, and salt tolerance.

In Arabidopsis, AtbHLH112 functions as a transcriptional activator that regulates genes involved in stress tolerance by binding to E-box motifs and a novel GCG-box motif (with the sequence 'GG[GT]CC[GT][GA][TA]C') . In peanut, AhbHLH112 improves drought tolerance by enhancing ROS-scavenging abilities and participating in ABA-dependent stress response pathways . In Artemisia annua, AabHLH112 is induced by low temperature and promotes artemisinin biosynthesis by regulating AaERF1 .

What specific targets do bHLH112 antibodies recognize across different plant species?

bHLH112 antibodies target epitopes within the bHLH112 protein, which contains a typical bHLH domain (approximately 50-60 amino acids). While the bHLH domain is highly conserved across species, the complete protein sequences show variation. For instance, in peanut, AhbHLH112 is 447 amino acids with a molecular mass of 48.7 kDa and an isoelectric point of 4.54 . The bHLH domain in AhbHLH112 spans amino acids 323-373 .

How can I verify the specificity of a bHLH112 antibody before experimental use?

To verify antibody specificity:

• Western blot analysis using recombinant bHLH112 protein as a positive control

• Comparative Western blots with wild-type and bHLH112 knockout/knockdown plant tissues

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Pre-adsorption tests using the recombinant antigen to block specific binding

• Cross-reactivity testing against related bHLH family members, particularly those in subgroup 12

When validating for cross-species applications, consider that AhbHLH112, AabHLH112, and AtbHLH112 share significant sequence homology but may contain unique epitopes. For example, phylogenetic analysis shows that AabHLH112 and AtbHLH112 cluster with Arabidopsis ICE proteins , suggesting shared epitopes that might affect antibody specificity.

What are the optimal fixation and sample preparation methods for immunolocalization of bHLH112 in plant tissues?

Based on research with bHLH112 transcription factors, consider the following protocol:

• Fixation: Use 4% paraformaldehyde in phosphate buffer (pH 7.2) for 2-4 hours at room temperature or overnight at 4°C

• Permeabilization: Include 0.1-0.5% Triton X-100 to ensure nuclear penetration, as bHLH112 is primarily localized in the nucleus

• Antigen retrieval: Apply heat-induced epitope retrieval using citrate buffer (pH 6.0) to counteract fixation-induced epitope masking

• Blocking: Use 3-5% BSA or normal serum in PBS with 0.1% Tween-20 for 1-2 hours

Special considerations for bHLH112:

• Include protease and phosphatase inhibitors during extraction, as AhbHLH112 and AtbHLH112 show stress-induced nuclear localization that may involve post-translational modifications

• Perform fixation quickly after stress treatments, as subcellular localization of bHLH112 can change in response to stressors like drought, salt, and ABA

What control samples should be included when using bHLH112 antibodies in functional studies?

For rigorous experimental design, include:

• Positive controls:

  • Tissues with confirmed high expression of bHLH112 (e.g., leaves for AhbHLH112, glandular secretory trichomes for AabHLH112)

  • Stress-induced samples, as bHLH112 expression increases under drought, salt, or cold stress

  • Overexpression lines of bHLH112 for signal validation

• Negative controls:

  • Tissues with minimal bHLH112 expression

  • bHLH112 knockout/knockdown lines

  • Primary antibody omission control

  • Non-specific IgG control at the same concentration as the primary antibody

• Experimental validation controls:

  • Time course samples following stress application, as bHLH112 expression is dynamic (e.g., in peanut, expression patterns differ in leaves, roots, and stems under drought stress)

  • Wild-type vs. transgenic comparisons

How can I optimize immunoprecipitation protocols for bHLH112 protein-DNA interaction studies?

For chromatin immunoprecipitation (ChIP) studies investigating bHLH112 binding to target promoters:

• Crosslinking optimization: Use 1% formaldehyde for 10-15 minutes, as excessive crosslinking may mask the epitope recognized by the antibody

• Sonication parameters: Adjust to generate 200-500 bp DNA fragments

• Antibody concentration: Titrate between 2-10 μg per ChIP reaction

• Washing stringency: Include high-salt washes to reduce background

When designing primers for qPCR validation:

• Target E-box motifs (CANNTG) in promoters of stress-responsive genes

• For AtbHLH112 specifically, include primers for the novel GCG-box motif ('GG[GT]CC[GT][GA][TA]C')

• For AhbHLH112, include primers for peroxidase (POD) gene promoter regions, particularly the P1 and P2 regions that interact with AhbHLH112 in yeast one-hybrid assays

How can I differentiate between specific and non-specific signals when using bHLH112 antibodies?

To distinguish genuine bHLH112 signals:

• Expected localization pattern: Confirm nuclear localization, as demonstrated for AhbHLH112, AabHLH112, and AtbHLH112

• Molecular weight verification: Look for a band at approximately 48-50 kDa (e.g., AhbHLH112 is 48.7 kDa)

• Expression pattern consistency: Verify that signal intensity increases under stress conditions (drought, salt, cold) that are known to induce bHLH112

• Comparative analysis: Compare wild-type vs. bHLH112 overexpression/knockout lines

• Competition assays: Pre-incubate antibody with recombinant bHLH112 to block specific binding

When using antibodies across species, consider that despite sequence homology, there may be species-specific variations in molecular weight, post-translational modifications, and stress responses that affect antibody recognition.

What are common artifacts in bHLH112 immunostaining and how can they be addressed?

Common artifacts and solutions:

Note that bHLH112 expression varies by tissue type and stress condition. For example, in peanut, AhbHLH112 shows highest expression in leaves, followed by roots under normal conditions, but expression patterns change dynamically under drought stress .

Why might bHLH112 antibody signals differ between stressed and non-stressed plant samples?

bHLH112 proteins show significant regulation under stress conditions:

• Expression level changes: bHLH112 transcription is strongly induced by stresses. For example, AhbHLH112 expression dramatically increases in all tissues in response to drought stress, with tissue-specific temporal patterns .

• Subcellular localization shifts: AtbHLH112 nuclear localization is induced by salt, drought, and ABA treatments , potentially affecting epitope accessibility.

• Post-translational modifications: Stress may trigger modifications affecting antibody recognition. AtbHLH112 functions as a transcriptional activator , which often involves phosphorylation or other modifications.

• Protein-protein interactions: Stress-induced interactions with other proteins may mask epitopes or alter antibody accessibility.

• Protein stability changes: Stress conditions may alter protein turnover rates, affecting abundance.

To account for these variables, include appropriate time-course sampling after stress application and consider cellular fractionation to separately analyze nuclear and cytoplasmic compartments.

How can bHLH112 antibodies be used to investigate transcriptional complexes in stress response pathways?

Advanced applications include:

• Co-immunoprecipitation (Co-IP): Use bHLH112 antibodies to pull down protein complexes, followed by mass spectrometry to identify interaction partners under different stress conditions.

• Sequential ChIP (ChIP-reChIP): Employ this technique to identify genomic regions co-occupied by bHLH112 and other transcription factors, revealing cooperative regulation mechanisms.

• Proximity ligation assay (PLA): Detect in situ protein-protein interactions between bHLH112 and potential partners identified through genetics or proteomics.

• Chromatin interaction analysis (ChIA-PET): Combine ChIP with chromatin conformation capture to identify long-range chromatin interactions mediated by bHLH112.

Research has shown that bHLH112 functions within complex regulatory networks. For example, AabHLH112 promotes artemisinin biosynthesis by binding to the AaERF1 promoter and enhancing its expression, which then directly activates artemisinin biosynthesis genes . Similarly, AtbHLH112 increases expression of P5CS genes while reducing expression of P5CDH and ProDH genes to increase proline levels during stress .

What methodological approaches can resolve conflicting data between transcript levels and protein abundance of bHLH112?

To address transcript-protein discrepancies:

• Parallel analysis: Simultaneously measure mRNA (RT-qPCR) and protein (Western blot) levels across multiple time points after stress application.

• Protein half-life determination: Use cycloheximide chase assays to assess bHLH112 protein stability under different conditions.

• Translational efficiency assessment: Employ polysome profiling to determine if bHLH112 mRNA translation is regulated post-transcriptionally.

• Proteasome inhibition: Compare protein levels with and without proteasome inhibitors to assess degradation contribution.

• Post-translational modification analysis: Use phospho-specific antibodies or mass spectrometry to identify modifications that might affect protein function without changing abundance.

Consider that bHLH112 regulation may differ between species and stress conditions. For instance, while AhbHLH112 expression increases under drought stress in all peanut tissues examined, the temporal patterns differ between leaves, roots, and stems .

How can bHLH112 antibodies be utilized in multiplexed imaging to understand spatial coordination of stress responses?

For multiplexed imaging approaches:

• Antibody labeling optimization:

  • Directly conjugate bHLH112 antibodies with fluorophores with minimal spectral overlap with other reporters

  • Use secondary antibodies with distinct fluorophores for multi-color imaging

  • Consider sequential immunostaining with antibody stripping between rounds for highly multiplexed approaches

• Co-localization studies:

  • Combine bHLH112 antibodies with markers for:

    • Other transcription factors (e.g., ERF1 in Artemisia annua)

    • Stress-responsive organelles (peroxisomes, chloroplasts)

    • RNA polymerase II to assess active transcription sites

• Spatial analysis across tissues:

  • Map bHLH112 distribution in relation to stress-responsive tissues

  • Correlate with physiological measurements (e.g., ROS accumulation, which is lower in AhbHLH112 overexpression lines)

• Time-course imaging:

  • Track dynamic changes in bHLH112 localization and abundance following stress application

  • Correlate with expression of target genes like POD, SOD, and stress-responsive genes

What cross-reactivity should be expected when using antibodies developed against one species' bHLH112 in another species?

Cross-reactivity considerations:

• Domain conservation: The bHLH domain (amino acids 323-373 in AhbHLH112) is highly conserved across species, suggesting potential cross-reactivity of antibodies targeting this region.

• Phylogenetic relationships:

  • AhbHLH112 (peanut) clusters with AtbHLH112 (Arabidopsis) in bHLH subgroup 12

  • AabHLH112 (Artemisia) clusters with Arabidopsis ICE proteins and AtbHLH112

  • This clustering suggests potential cross-reactivity among these species

• Epitope mapping: Determine which protein region the antibody recognizes:

  • Antibodies against the bHLH domain: Higher cross-reactivity expected

  • Antibodies against N/C-terminal regions: Lower cross-reactivity likely

  • Consider that AhbHLH112 has a sequence variation at site 951 (C instead of G) between different peanut genome databases

• Validation requirements:

  • Always perform Western blot validation when using antibodies across species

  • Include positive controls from the original species

  • Consider testing with recombinant proteins from target species

How do post-translational modifications of bHLH112 differ between species and how might this affect antibody recognition?

Post-translational modification considerations:

• Nuclear localization regulation: AtbHLH112 nuclear localization is induced by salt, drought, and ABA , suggesting potential phosphorylation or other modifications that might affect antibody binding.

• Functional domains:

  • The N-terminus of AtbHLH112 contains the activation domain

  • Modifications in this region might be important for transcriptional activity

  • Antibodies targeting modified regions may show differential binding based on activation state

• Species-specific regulation:

  • AabHLH112 is induced by cold stress

  • AhbHLH112 is induced by drought stress

  • AtbHLH112 responds to salt, drought, and ABA

  • These different stress responses may involve species-specific post-translational modifications

• Experimental approach:

  • Use phospho-specific antibodies if phosphorylation sites are known

  • Consider 2D gel electrophoresis to separate differently modified forms

  • Use mass spectrometry to identify and compare modifications across species

What comparative binding assays can determine if bHLH112 proteins from different species recognize the same DNA motifs?

To investigate DNA-binding conservation:

• Electrophoretic mobility shift assay (EMSA):

  • Compare binding of bHLH112 proteins from different species to:

    • E-box motifs (CANNTG), the canonical bHLH binding site

    • GCG-box motif ('GG[GT]CC[GT][GA][TA]C'), specifically bound by AtbHLH112

    • Promoter fragments of common target genes (e.g., POD, SOD)

• Chromatin immunoprecipitation (ChIP):

  • Use bHLH112 antibodies to immunoprecipitate chromatin from different species

  • Perform ChIP-seq to globally compare binding sites

  • Focus analysis on orthologous genes to assess conservation of regulation

• Yeast one-hybrid (Y1H):

  • Test binding of different bHLH112 proteins to:

    • The POD promoter (as AhbHLH112 binds to P1 and P2 regions)

    • The ERF1 promoter (bound by AabHLH112)

    • Promoters of stress-responsive genes

• Dual-luciferase assays:

  • Compare the ability of bHLH112 proteins from different species to activate:

    • Stress-responsive gene promoters

    • The ERF1 promoter (activated by AabHLH112)

    • Promoters containing E-box or GCG-box motifs

Research has shown different binding preferences: AhbHLH112 interacts with the POD promoter , AabHLH112 binds to the ERF1 promoter but not directly to artemisinin biosynthesis gene promoters , and AtbHLH112 binds both E-box and GCG-box motifs .

What emerging techniques could enhance the specificity and application range of bHLH112 antibodies?

Emerging technologies to consider:

• CRISPR epitope tagging: Add small epitope tags to endogenous bHLH112 genes, allowing use of highly specific commercial tag antibodies while maintaining native expression patterns and regulation.

• Nanobodies: Develop single-domain antibodies against bHLH112, which offer advantages including smaller size, better tissue penetration, and potential for intracellular expression.

• Proximity-dependent labeling: Use bHLH112 antibodies coupled with enzymes like BioID or APEX2 to identify proximal proteins in living cells, revealing the dynamic bHLH112 interactome during stress responses.

• Single-molecule imaging: Apply super-resolution microscopy techniques with highly specific bHLH112 antibodies to track individual molecules during stress responses.

• Intrabodies: Develop antibody fragments that function within living cells to track or modulate bHLH112 activity in real-time during stress responses.